The alternative packaging of DNA into euchromatic and heterochromatic forms can be stably maintained in differentiated cells, and represents an important form of epigenetic inheritance. Malfunction in the heterochromatin system can lead to misregulation of gene expression, as well as chromosome instability, and is a contributing factor in many health problems, including cancer and genetically-based developmental disabilities. Heterochromatin packaging can be perpetuated and spread by virtue of an interacting set of structural proteins, with Heterochromatin Protein 1 (HP1a) playing a key role in both recognizing a critical histone modification mark (methylation of H3K9) and interacting with the histone methyl transferase (HMT). Our long term goal is to understand why and how certain regions of the genome are selected for heterochromatin assembly, and to identify the required chromosomal proteins and their interactions. All work will be carried out in Drosophila, a model system that allows genetic, cytological, and biochemical approaches;position effect variegation (PEV) is used as an indicator of heterochromatin packaging. Our studies of chromosome four (entirely heterochromatic by several measures) indicate that remnants of the 1360 DNA transposon serve as targets for heterochromatin formation, but that stable heterochromatin requires a high repeat density. In the first specific aim, the role of genome organization and identity of specific targets will be tested using reporter P elements with different types and copy number of repetitious elements. In our second aim, binding studies and structural analysis will explore how HP1a discriminates among its several partners to assemble a silencing structure. The HMT specific for chromosome four will be identified, its pattern of interactions explored, and the impact on spreading considered. Additional heterochromatin components will be identified by screening for proteins interacting with HP2 in a yeast two hybrid system exploiting recently recovered HP2 mutations as controls. In a third long-term aim, we will ask whether some fourth chromosome genes function optimally in a heterochromatic environment, and explore that adaptation. Taken together these studies will illuminate how heterochromatin formation is initiated and sustained, and will provide new insights into gene regulation at the domain level.